Journal of Applied Phycology 10: 383389, 1998.

1998 Kluwer Academic Publishers. Printed in the Netherlands.

383

Nitrogen availability influences the biochemical composition and

photosynthesis of tank-cultivated Ulva rigida (Chlorophyta)
Juan Luis Gomez Pinchetti , Elena del Campo Fernandez, Paula Moreno Dez & Guillermo
Garca Reina
Instituto de Algologa Aplicada, Universidad de Las Palmas de G.C., Muelle de Taliarte s/n, 35214 Telde, Las
Palmas, Canary Islands, Spain
( Author for correspondence; fax: +34 928 132830; e-mail: pincheti@infovia.ulpgc.es)
Received 13 March 1998; revised 25 May; accepted 25 May 1998

Key words: ammonium, C:N ratio, tank culture, dietary fibre, fatty acids, nitrogen, photosynthesis, Ulva rigida

Abstract
Physiological and biochemical changes in relation to inorganic nitrogen availability were studied for tank-cultivated
Ulva rigida grown under nitrogen- enriched and nitrogen-depleted seawater. U. rigida was initially cultivated in
nitrogen-enriched seawater (daily concentrations of NH4 + and NO3 + NO2 ranged between 0.51.7 and 0.06
0.15 mg L1 , respectively), then transferred to nitrogen-depleted seawater where photosynthetic capacity decreased
to zero after 23 d. At the time (14 d) when photosynthetic rates were lower than 2.0 mol O2 g1 FW min1
and strong bleaching had occurred, some algae were returned to the initial nitrogen-enriched seawater to study
recovery from N-limited growth. Data on biochemical composition (chlorophylls, ash, caloric content, fatty acids
and dietary fibres) and colouration varied significantly depending on the nitrogen conditions. C:N ratios correlated
significantly with biochemical parameters. Fatty acid (FA) synthesis continued during the N-starvation period;
saturated and mono-unsaturated FA increased to a maximun of 72.2%, while poly-unsaturated fatty acids (PUFA)
decreased to 27.7%. During the N-enriched recovery period, the reverse was found. C:N ratios above 10 correlated
with carbohydrate synthesis as shown by the dietary fibre level. Under nitrogen enriched conditions, C:N ratios
decreased along with a decrease in fibre level. Under controlled conditions, nitrogen represents a major influence
on the development of intensive tank cultivation of Ulva rigida, not only by affecting parameters closely related to
nitrogen metabolism but also some clearly influenced by carbon uptake.

Introduction
Interest in seaweeds as novel foods with potential
nutritional benefits (Darcy-Vrillon, 1993), fodder (recently for fish culture purposes; Davies et al., 1997),
fertilizers and sources of fine chemicals for the pharmaceutical, food and chemical industries is expanding
in Western countries. In fact, cultivation techniques
are being improved with the main objective of obtaining higher algal biomass that exhibit specific qualities
(Lobban & Harrison, 1994).
Physical and chemical factors such as temperature,
salinity and light (Lobban & Harrison, 1994), aera-

tion (Chen & Johns, 1991) or nutrient concentrations

(Bjrnster & Wheeler, 1990; Floreto et al., 1996;
Garca-Ferris et al., 1996) influence the biochemical
composition, physiological status and ultrastructure of
micro- and macroalgae. Nitrogen is one of the most
important limiting nutrients in the marine environment
(Hanisak, 1983) and nitrogen control is critical for the
intensive cultivation of algae due to its role in growth,
and regulation of metabolism (Smit et al. 1997).
The green macroalga Ulva has been widely used
as a biofilter because of its high efficiency to remove nitrogenous inorganic compounds (up to 90%

384
in the form of ammonium) from wastewaters (Neori et al., 1991; Jimnez del Ro et al., 1996).
Ulva species show the capacity to utilize, quickly
absorb and metabolize different forms of inorganic
nitrogen, mainly nitrate and ammonium, depending
on their availability. However, ammonium (NH4 + ),
which can be toxic or inhibitory for some seaweeds
at concentrations higher than 3050 m, is the preferred nitrogen form for Ulva and other species of
macroalgae (Lobban & Harrison, 1994). Moreover,
Jimnez del Rio et al. (1995) demonstrated that
NH4 + availability controls ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) activities in Ulva rigida, confirming that
there is a close relationship between inorganic nitrogen, photosynthesis and carbon metabolism (see
review by Turpin, 1991).
Excess and depletion of nitrogen sources in the
culture medium causes important cellular responses
in algae. Excess nitrogen, as well as other nutrients,
can be stored and used for growth during nutrient
limited periods by a number of macroalgae including
Ulva fenestrata and Enteromorpha intestinalis (Bjrnster & Wheeler, 1990), Codium fragile (Hanisak,
1979), Chaetomorpha linum (McGlathery et al., 1996)
or Gracilaria spp. (Jones et al., 1996; Smit et al.,
1997). Storage occurs as inorganic nitrogen (NO3
and NH4 + ) (Chapman & Craigie, 1977) and organic
compounds, amino acids and proteins (Jones et al.,
1996; McGlathery et al., 1996). When nitrogen limits growth, carbohydrate synthesis predominates and
accounts for the increase in polysaccharide levels and
related high C:N ratio values (Vergara et al., 1993;
Lahaye et al., 1995).
Few reports have specifically considered the effects of nitrogen resupply on photosynthesis, algal
recovery and biochemical transformation of seaweeds.
The study of these effects can clarify processes by
which nutrient resupply enhances growth and nutrient
utilization. The present study investigates the influence of N-limitation and N-enrichment on growth and
a number of physiological and biochemical characteristics of tank-cultivated Ulva rigida, and in particular,
evaluates how nitrogen supply can alter and/or modify
specific properties of interest in the composition and
physical properties of the biomass.

Materials and methods

Algal material and cultivation conditions
Ulva rigida C. Agardh was collected from Taliarte
harbour, east coast of Gran Canaria (Canary Islands)
and cultivated in 750 L aerated tanks at a density of
2.5 g L1 . Nitrogen enriched seawater at turnover
rates of 8 vol d1 was pumped from a 2000-m3
tank with approximately 40 t of gilthead seabream
(Sparus aurata). Daily concentrations of NH4 + and
NO3 + NO2 ranged between 0.51.7 and 0.06
0.15 mg L1 , respectively. Maximum irradiance levels
were 1975 98 mol photon m2 s1 and the water
temperature ranged between 20 and 24 C. Relative
growth rates (% d1 ) were calculated according
to the equation = 100 ln (Wt /W0 )/t (DElia & Deboer, 1978), where W0 = initial fresh weight (FW),
Wt = final fresh weight, and t = time in days.
After one month, algae were transferred (under the
same physical conditions) to running seawater without
any nitrogen addition (NH4 + + NO3 + NO2 concentrations 3 m). After 14 d, when photosynthetic
rates reached values lower than 2.0 mol O2 g1 FW
min1 and strong bleaching was observed, algae were
returned (early in the morning) to the initial nitrogen
enriched seawater to study recovery.
Prior to analysis samples were washed with distilled water and freeze dried. Determination of C:N
ratios was performed with a Perkin Elmer Elemental
Analyzer Model 2400 CHN. Caloric content values
were determined by combustion in an Ika-calorimeter
C700 (Janke & Kunkel). Percent ash was determined
by combusting dried samples at 450 C to a constant
weight. Determinations were carried out in triplicate.
Where not indicated, variability did not exceed 10%
of the mean.
Photosynthesis and chlorophyll measurements
Oxygen exchange was measured with a Clark-type
oxygen electrode fitted with a water jacketed chamber
(Hansatech Instruments Ltd., UK) at 20 C. Thallus
fragments (10 mg FW) were transferred to the chamber containing 2 mL filtered (through 0.2 m filters)
seawater buffered at pH 8.2 with 30 mm TRIS. Light
was provided by a slide projector (Reflecta, Germany)
and irradiance measured with a LI-1000 data logger
and a spherical quantum sensor LI-193SA (Li-Cor,
USA). Oxygen evolution at a Photon Flux Density
(PFD) of 750 mol photon m2 s1 was recorded.

385

Figure 1. Maximum photosynthetic rates of nitrogen-starved

(crossed bars) and nitrogen-enriched (black bars) Ulva rigida.
Arrow shows the day of change of nitrogen-starved algae to nitrogen-enriched seawater. Measurements were taken at 750 mol
photon m2 s1 and 20 C.

Figure 2. Effect of nitrogen starvation ( ) and enrichment ( ) on

the C:N ratio of Ulva rigida. Arrow shows the day of change of
nitrogen-starved algae to nitrogen-enriched seawater. Bar shows the
C:N value of wild collected U. rigida.

Table 1. Effect of N-starvation and N-enrichment (indicated as N and + N, respectively) on the chlorophyll a
and b concentrations of tank-cultivated Ulva rigida. The
starting day of the nitrogen starvation period is indicated
as day 0. Algal recovery in N-enriched seawater started on
day 14, independent of tanks where N-starved U. rigida
was maintained to day 23 (as indicated in Figure 1). Mean
SD; n = 5.
Time Chl a
Chl b
Chl a/Chl b
(day) (g g1 FW) (g g1 FW) ratio
N

0
15
21
+N 14
16
20

1162.5 26.2
54.6 6.7
40.0 7.1
65.4 7.0
381.7 54.3
672.2 70.2

604.9 29.9
29.9 4.2
20.3 3.2
43.9 4.6
281.5 28.3
418.3 60.1

1.92
1.83
1.97
1.49
1.35
1.61

Chlorophylls a and b were extracted in 90%

acetone and determined according to Jeffrey and
Humphrey (1975).
Fatty acids and dietary fibre analysis
Lipid extraction and trans-esterification of freeze dried
and ground samples (particle size < 200 m) were
carried out as described by Cohen et al. (1987). Fatty
acid methyl esters (FAME) were separated by capillary gas chromatography using a Shimadzu GC-15A
equipped with FID and a Shimadzu Chromatopac
C-R5A integrator. Samples (1.0 L) were injected
in a Supelcowax 10 fused silica capillary column
(30 m 0.32 mm) using helium as the carrier gas

Figure 3. Effect of nitrogen starvation ( ash; caloric content)

and enrichment ( ash; caloric content) on the ash and caloric
contents of Ulva rigida. Arrow shows the day of change of nitrogen-starved algae to nitrogen- enriched seawater. Bars show ash
(black bar) and caloric contents (crossed bar) of wild collected U.
rigida.

and operated at 180 C during 10 min and then at

215 C after an increase rate of 2.5 C min1 . Both
the injector and detector temperature was 250 C.
For quantitative analysis, nonadecanoic acid (19:0)
methyl ester was added as an internal standard. Peaks
were identified by comparison with known standards
(Sigma and Supelco).
Soluble and insoluble dietary fibre content of
freeze dried samples were determined as described
by Lahaye (1991). Percent of soluble and insoluble dietary fibre was obtained by subtracting total
residual proteins and ash contents from the respective
yield of the liquid and solid fractions. Proteins were

386
Table 2. Effect of N-starvation and N-enrichment (indicated as N and + N, respectively) on the fatty acid
composition (% total fatty acids) of tank-cultivated Ulva rigida. The starting day of the nitrogen starvation
period is indicated as day 0. Algal recovery in N-enriched seawater started on day 14. Wild; fatty acids
composition from wild collected U. rigida.
N
Day
0

12

18

14

16

22

3.3
47.3
1.2

3.3
31.7
0.4

2.9
38.7
0.4

1.5
49.9

58.1

3.1
47.5

1.8
48.1
0.7

1.7
31.3
0.6

2.9
1.9

2.3
16.8

0.3
0.5
3.5
0.6
8.1

2.9
2.5
1.8
10.7

14.2

0.5
1.5
0.6
9.7

2.5
12.1

1.1
2.7
1.6
10.9

0.2
0.6
3.1
1.0
5.5

5.0

7.1

4.7

4.9

4.9

5.4

5.8

5.4

7.5

13.3

14.1

10.9

11.8

11.5

11.3

1.0
16.5
0.2
0.8

2.7
3.1
0.2

10.6
14.3

0.3
0.6

9.0
12.3

0.5

4.5
6.9

6.7

4.3
6.6

2.8
6.2
0.7

0.8

11.1
13.9
0.9
0.3
0.8

4.4
1.5
75.4
24.4

0.3
2.4
2.7
48.4
51.6

3.5

69.3
30.7

4.3

72.3
27.7

0.5
2.5
2.6
66.9
33.2

0.5
1.7
3.1
44.0
56.2

Wild

Saturated
14:0
16:0
18:0
Mono-unsaturated
16:1w9
16:1w7
16:1w5
18:1w9
18:1w7
Di-unsaturated
18:2w6
Tri-unsaturated
18:3w6
18:3w3
20:3w6
22:3w6
Tetra-unsaturated
16:4w1
18:4w3
18:4w1
20:4w6
20:4w3
Penta-unsaturated
20:5w6
20:5w3
22:5w3
Total S & M
Total PUFA

+N

3.1
2.1
54.3
45.8

determined as nitrogen using the Kjeldahl method

(N 6.25).
Results
Photosynthesis and pigment composition
In the initial nitrogen enriched condition, Ulva rigida
growth rates ranged between 12 and 16% d1 , chlorophyll concentration reached maximum levels (Table 1)
and algae became a deeply pigmented green. Maximum photosynthetic rates reached 6.8 mol O2 g1
FW min1 (Figure 1).

1.7

5.4

66.9
33.2

When transferred to nitrogen depleted seawater,

growth rates decreased to less than 2% d1 and material ceased growing after 5 d. Algae became bleaching and photosynthetic rates decreased significantly
to zero on day 23 (Figure 1). With N-enrichment
renewal for some algae on day 14, there was a recovery of chlorophyll, resulting in greater pigmentation,
and photosynthesis (Table 1, Figure 1). Following Nenrichment, algae reached maximum photosynthetic
levels that were higher than the initial material within
6 d. Chlorophyll levels paralleled the variations in
photosynthetic rates and chlorophyll a/b ratios were

387
constant, independently of the nitrogen conditions
(Table 1).
C:N ratios
Lower C:N values were observed in algae grown in
nitrogen enriched seawater and increased until day
18 when values approached 35, indicating nitrogen
limitation (Figure 2). Freshly collected algae showed
intermediate values suggesting that they were nitrogen
limited. Maximum and minimum carbon tissue composition ranged from 36.4% measured on the starting
day of nitrogen starvation period (indicated as day 0)
to 15.7% on day 16 of the nitrogen enrichment period.
Nitrogen tissue values varied from 5.7% on day 20 of
the nitrogen enrichment period, to 0.6%, on day 18 of
nitrogen starvation. In wild algae, the percent of tissue
carbon and nitrogen were 28.7 and 1.3, respectively.
Ash and caloric content values
Ash and caloric contents in algae varied inversely with
N conditions (Figure 3); nitrogen limited algae had
greater ash content and less caloric content. Wild algae
showed intermediate values for both variables.
Fatty acids and dietary fibre contents
Total saturated and mono-unsaturated FA increased
and total poly-unsaturated FAs decreased during nitrogen starvation to reach values close to those found
in wild algae (Table 2). During the acute N-limitation
period, fatty acids were represented mainly by six FAs
(16:0; 18:1w7; 18:2w6; 18:3w3; 18:4w3 and 20:5w3).
When returned to nitrogen enriched conditions the FA
composition was reversed; total saturated and monounsaturated FA decreased and PUFA increased. During this recovery period, PUFA not found in wild algae
appeared (18.3w6, 20.3w6, 22.3w6, 20.4w6, 20.4w3,
20.5w6), but were not major constituents.
Major FAs were palmitic (16.0) and vaccenic
(18.1w7) acids. Both increased, from 31.8 to 58.0%
and 8.1 to 14.2% of total FA, respectively, during starvation. The remaining FAs disappeared at the peak
of nitrogen starvation and reappeared under nitrogen enriched conditions. In general, PUFA decreased
in algae grown in nitrogen starved conditions except for eicosapentaenoic acid (20.5w3) which showed
a transitory increase. Major PUFAs were linolenic
(18.3w3), parinaric (18.4w3) and hexadecatetraenoic
(16.4w1) acids.

A general increase in dietary fibre was observed

in nitrogen starved algae, with a decrease during the
recovery period (Table 3). The soluble fibre fraction
was higher compared to the insoluble fraction.

Discussion
The biochemical and physiological characterization of
nitrogen enrichment and starvation in previous studies has demonstrated how macroalgae may respond
to a temporal imbalance of N availability (McGlathery et al., 1996). Further, the N requirements for
growth, storage and use during subsequent periods of
low external nutrient supply have been described (Lapointe & Ryther, 1979; Bjrnster & Wheeler, 1990;
McGlathery et al., 1996).
In tank-cultivated Ulva rigida, decrease in growth
and bleaching occurred during the nitrogen starvation
period. Nitrogen previously stored in algae is used
in growth when N becomes limiting or when light
or temperature increase (Duke et al., 1986; Coutinho
& Zingmark, 1993). Bleaching, a result of pigments
being metabolized as a source of protein, has also
been described for Gracilaria (Smit et al., 1997). A
change in the ratio of Chl a to accessory chlorophyll
was evident for Chl a/b ratios during N-enrichment
and starvation in Ulva, and is a common phenomenon
(Turpin, 1991). Shifts in chlorophylls content were
important even though daily irradiance values reached
high maximum levels (up to 1900 mol photon m2
s1 ) suggesting that pigment content was controlled
by dissolved nitrogen and not by light.
The photosynthetic capacity during nitrogen starvation progressively declined to zero after 23 d,
while algae showing photosynthetic rates of 2.0 mol
O2 g1 FW min1 and transferred to nitrogen
enriched conditions recovered rapidly. Nitrogendeficiency clearly affects the light harvesting system
and the activity of the enzymes of inorganic carbon
metabolism (Turpin, 1991; Jimnez del Rio et al.,
1995), in addition to growth. It appears that soluble
proteins decrease under low nitrogen levels in relation
to the internal nitrogen pool (Turpin, 1991).
The C:N ratio is a strong index of the physiological
status of micro- and macroalgae (Chen & Johns, 1991;
Vergara et al., 1993). The ratio shows low values when
N was abundant and increased when N supply was
limited. C:N values close to 10 have been described
as optimal or normal for the nitrogen status of algae
and a ratio greater than 10 indicates N-limitation in

388
Table 3. Effect of N-starvation and N-enrichment (indicated as N and + N, respectively) on the soluble,
insoluble and total dietary fibres composition (%) of
tank-cultivated Ulva rigida. The starting day of the
nitrogen starvation period is indicated as day 0. Algal recovery in N-enriched seawater started on day
14. Wild; digestive fibres composition from wild
collected U. rigida.
Wild

Soluble
Insoluble
Total

30.6
6.3
36.9

N
Days
0
6

+N
18

14

20

34.6 35.1 37.1 37.0 36.2

8.8 13.9 12.3 9.7 7.7
43.4 49.0 49.4 46.7 43.9

Gracilaria (Lapointe & Ryther, 1979). In the present

study, freshly collected Ulva rigida was nitrogen deficient as indicated by its C:N value close to 22. During
the experiments, stored N was used to maintain algae
at least for 23 d under N-depleted seawater conditions.
Ash content correlates with C:N ratio suggesting
control by nitrogen uptake and influencing the organic
and caloric content of Gracilaria (Lapointe & Ryther,
1979). In our study, N-starved Ulva rigida (higher
C:N ratios) showed a higher ash content and lower
caloric values than N-enriched algae (lower C:N values). These findings contrast with those of Lapointe
& Ryther (1979) who found that N-limited Gracilaria
contained less ash and a higher caloric content per
unit dry weight; however, caloric content of the ashfree organic matter was higher in N-enriched algae.
Also, on an ash-free basis, low caloric value and high
ash content coincided with maximal growth of several
freshly collected red and brown algae (Himmelman &
Carefoot, 1975).
During the experiments, nitrogen availability also
affected levels of protein, amino acids, (both decreasing in nitrogen limited conditions; Betancort, 1997)
and carbohydrate. The dietary fibre content found was
similar to that obtained previously by Lahaye et al.
(1995). In U. rigida, when nitrogen limits growth,
carbohydrate synthesis predominates as shown by the
dietary fibres content whereas, in nitrogen enriched
conditions, a decrease in fibres composition is observed.
Fatty acids synthesis continued during N-starvation
with saturated and mono-unsaturated FAs increasing
to a maximun, while PUFA decreased. The increase
in specific FAs might be related to the formation of

lipid layers which play a role as light filters preventing

photooxidative damage and in reducing water losses,
as for Chlorella zofingiensis (Bar et al., 1995).
FA composition reversed during the N-enriched
recovery period suggesting that PUFAs are used to create storage compounds in optimum conditions which
can then be used as energy sources during periods of
stress. During recovery, FAs which were not present in
wild algae or were minority components in N-starved
Ulva, may be used by intermediary precursors for the
group of major fatty acids.
All results correlate with changes in the C:N ratio.
Chen and Johns (1991) indicated that the proportion of
unsaturated FAs (especially trienoic acids) increased
significantly when C:N ratios were low in Chlorella
sorokiniana. At higher C:N ratios, carbon is supplied
in excess, nitrogen becomes limiting, and the increase
of the cell lipid content may be explained as a carbon
storage mechanism. Similar results were described for
Gracilaria verrucosa by Pohl and Zurheide (1979).
Our results suggest that, under controlled conditions, nitrogen (mainly in the form of NH4 + ) presents
a strong influence on the development and composition of intensive tank-cultivated Ulva rigida, not
only by affecting parameters closely related to nitrogen metabolism but also to carbon uptake. Data on
nitrogen requirements for growth, storage and use during limitation and resupply periods agree with those
previously described for macroalgae. However, it is
demonstrated that nitrogen availability clearly affected
the specific composition of important biochemical
parameters such as chlorophylls, saturated and unsaturated fatty acids, dietary fibres, ash and caloric
content, which were directly correlated with C:N ratio
dynamic.

Acknowledgements
The authors are indebted to Lidia Robaina, Mara
Salhi and H. Fernndez-Palacios (GIA, Instituto Canario de Ciencias Marinas, ICCM) for helpful advice
during the experiments. We thank Dr B.A. Whitton,
Dr M.T. Brown (Department of Biological Sciences,
University of Plymouth) and an anonymous reviewer
for helpful comments on the manuscript.

389
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